The efficacy of DNA crosslinking cancer chemotherapeutics relies on many factors including the ability for the DNA to be repaired. This project focuses on the mechanism of repair of synthetic CNG 1,3 interstrand DNA crosslinks as a model for clinically relevant products of prescribed DNA crosslinking agents. The project employs the use of a fast DNA repair assay to study the repair of the crosslinks in mammalian cell-free extracts by Nucleotide Excision Repair (NER). These experiments are conducted on DNA that has been adsorbed onto a 96 well plate; repair is detected using a biotin/streptavidin-fluorophore system. Also studied is the affinity of individual NER factors for the crosslinked substrate. These are examined by electrophoretic mobility shift assays or a modification of the plate assay using protein-specific antibodies. Complementary structural studies involving the use of chemical probes are conducted to determine the amount of conformational or other changes induced by an interstrand crosslink. The results of this project should provide valuable insights into the mechanism of DNA repair and ways of circumventing the problem of innate or acquired resistance to DNA crosslinking agents as well as potential inhibitors to pathways of repair.